Insulin’s Regulation of c-fos Gene Transcription in Hepatoma Cells*

In H4IIE rat hepatoma cells insulin interacts with its receptors to induce DNA synthesis and promote cell division. However, the postreceptor events that lead to DNA synthesis and cell division have not been well characterized. Previous studies indicate that insulin can regulate the expression of several genes in H4 cells. One of these genes is the proto-oncogene c-fos, a cellular gene whose deregulation has been implicated in the process of cellular differentiation and division. In the present work insulin is shown to regulate cellular c-fos mRNA accumulation and the transcription rate of the c-fos gene. Insulin caused a rapid, dose-dependent increase in the cytoplasmic concentration of c-fos mRNA which was maximal by 30 min. Preceding this, a more rapid 6-8 fold increase in transcription of the c-fos gene was observed. Induction of transcription was apparent following only 5 min of insulin addition. This is the most rapid effect of insulin yet demonstrated on the induction of gene expression. Protein synthesis inhibitors (cycloheximide, anisomycin) also induced the transcription of the c-fos gene. However, they stimulated a much greater increase in transcription than did insulin, and followed a different time course of action. The addition of insulin in combination with a protein synthesis inhibitor resulted in no greater increase in c-fos transcription than did the addition of a protein synthesis inhibitor alone. The nonadditivity of H4 cell c-fos gene expression may indicate a similar mode of action by insulin and protein synthesis inhibitors.

In H4IIE rat hepatoma cells insulin interacts with its receptors to induce DNA synthesis and promote cell division.
However, the postreceptor events that lead to DNA synthesis and cell division have not been well characterized.
Previous studies indicate that insulin can regulate the expression of several genes in H4 cells. One of these genes is the proto-oncogene c-fos, a cellular gene whose deregulation has been implicated in the process of cellular differentiation and division. In the present work insulin is shown to regulate cellular c-fos mRNA accumulation and the transcription rate of the c-fos gene. Insulin caused a rapid, dose-dependent increase in the cytoplasmic concentration of c-fos mRNA which was maximal by 30 min. Preceding this, a more rapid 6-a-fold increase in transcription of the c-fos gene was observed.
Induction of transcription was apparent following only 5 min of insulin addition. This is the most rapid effect of insulin yet demonstrated on the induction of gene expression. Protein synthesis inhibitors (cycloheximide, anisomycin) also induced the transcription of the c-fos gene. However, they stimulated a much greater increase in transcription than did insulin, and followed a different time course of action.
The addition of insulin in combination with a protein synthesis inhibitor resulted in no greater increase in c-fos transcription than did the addition of a protein synthesis inhibitor alone. The nonadditivity of H4 cell c-fos gene expression may indicate a similar mode of action by insulin and protein synthesis inhibitors.
The proto-oncogene c-fos is one of the early response genes which is expressed in response to a variety of growth factors (l-9). The controlled expression of the normal cellular fos (c-fos) gene may be involved in the signaling mechanisms within the cell which lead to an increase in DNA synthesis and cell division. A rapid induction in the transcription of the fos gene was observed following growth factor or mitogen addition to quiescent fibroblasts, macrophages, myoblasts, and pheochromocytoma cells (for reviews see Refs. 10 and 11; also see Refs. 2,3,7,and 8 mals. When this hormone was deficient, or when cells were resistant to the actions of this hormone, normal growth was impaired (18,19). The ability of insulin to induce DNA synthesis was shown in a variety of cell types in culture including human fibroblasts, mouse 3T3 fibroblasts, mouse pre-adipocytes, hamster kidney cells, rat liver cells, and rat and human hepatoma cells (20)(21)(22)(23)(24)(25). Insulin was also found to regulate the c-fos gene in 3T3-Ll adipocytes (5). However, this effect of insulin on c-fos induction was not exclusive. Other growth factors, including platelet-derived growth factor, epidermal growth factor (EGF),' and bombesin, also increased accumulation of c-fos mRNA in 3T3 cells (1, 2, 6). Recently, insulin was shown to increase accumulation of c-fos mRNA in a rat hepatoma cell line (26).
The c-fos gene is one of a group of genes thought to be involved in events leading to cell division. Since rat H4IIE hepatoma cells divide in response to insulin, we hypothesized that one of the initial actions of insulin would be to induce transcription of the c-fos gene. In the experiments presented here, insulin was found to induce c-fos gene transcription as were protein synthesis inhibitors. Regulation of c-fos mRNA by Insulin-Insulin increased the concentration of c-fos mRNA in a time-and dose-dependent manner. As shown in Fig. 1, insulin (5 X lo-' M) stimulated the cytoplasmic accumulation of c-fos mRNA in H4 cells (lanes b, e, and h) compared to cells not treated with insulin (lanes a, c, and fl. Insulin increased c-fos mRNA levels approximately lo-fold and S-fold by 30 and 60 min, respectively, followed by a decrease to 3-fold by 120 min (Table I) centrations of insulin greater than 5 X lo-' M did not result in a further rise in c-fos mRNA levels and lower concentrations gave proportionally less induction (data not shown).

Transcriptional
Effects of Insulin on c-fos and Other Oncogenes-Several control experiments were performed to confirm the linearity of our nuclear run-off assays and have been presented previously (30). In the present studies, there was a small, but consistent Z-3-fold increase in c-fos transcription within 5 min of insulin addition (5 x lo-' M), reaching a maximum of 6-7-fold induction within 15 min (Fig. 2, A and B). This increase was short-lived since the induction declined to 4-fold in 30 min and returned to basal levels within 60-120 min. The effect of insulin on transcription of the c-fos gene was also determined at 24 h following insulin addition and was found to be comparable to transcription rates in basal, untreated cells (data not shown). Insulin had no effect on the transcription of the @-tubulin gene.
The ability of increasing concentrations of insulin to induce the transcription of c-fos mRNA is shown in Fig. 3. A minimal concentration of 5 X lo-" M insulin was necessary to obtain a 75% increase in transcription of c-fos compared to control values, with 5 X lo-' M insulin resulting in a maximum 7-fold stimulation. Higher concentrations of insulin resulted in no H4 cells were deprived of serum for 24 h and treated with insulin (5 x lo-' M). Nuclei were then isolated by sucrose density ultracentrifugation.
Transcription was measured using a nuclear run-off assay in which the isolated nuclei were incubated in the uresence of I"*PlUTP and the labeled RNA was hybridized to C-/OS or @-tubulin EDN'As that were immobilized on nitrocellulose as described under "Experimental Procedures." The filters were washed extensively and autoradiographed (A). further increase in c-fos transcription.
The transcription of the P-tubulin gene was not affected by any concentration of insulin.
The effects of insulin on the transcription of several other oncogenes were studied and insulin was found to have no effect on transcription of the sis, ~53, ros, erb-b, and myb oncogenes (data not shown).
Transcriptional Effects of Serum and Other Growth Factors on the c-fos Gene-Fetal bovine serum has been shown to stimulate cell division of H4 cells (21). Since the c-fos gene is a growth-related gene, the effect of serum on the regulation of transcription of this gene was determined. The addition of 5 or 10% fetal bovine serum to 24-h serum-starved H4 cells resulted in a 6-or &fold increase in c-fos transcription, respectively (Fig. 4, serum lanes b and c). The induction of c-fos returned to basal transcription rates by 120 min following serum administration (Fig. 4, serum lanes d and e). This time course was similar to that observed with insulin treatment.
Two other growth factors, EGF and FGF, were ineffective in altering the transcription rate of the c-fos gene when added individually to serum-deprived H4 cells (Fig. 4, EGF-FGF  lanes b and c). Likewise, transcription of the sis, ~53, ros, erb-b, and myb oncogenes did not respond to the addition of serum, EGF, or FGF under the conditions tested (data not shown).

DISCUSSION
Growth factors such as EGF, platelet-derived growth factor, and FGF play important roles in the stimulation of DNA synthesis and cell division (11). The effects of insulin as a growth factor, however, have been poorly studied. Since insulin can interact with receptors for other growth factors, it has been difficult to determine if insulin's growth stimulatory effects are mediated through its own receptor or through other growth factor receptors. In the rat H4IIE hepatoma cell line used in the current experiments the growth-promoting effects of insulin were due solely to insulin's interaction with its own receptor (37-39). Insulin has been shown to regulate the expression of several genes in rat hepatoma cells. For example, insulin stimulates the transcription of the p33 and glyceraldehyde-3-phosphate dehydrogenase genes (40, 41) while it inhibits the transcription of the phosphoenolpyruvate carboxykinase gene (42,43). In 3T3-Ll adipocytes insulin induced the accumulation of cfos mRNA (5). However, the effects of insulin were not exclusive, since other growth factors, including platelet-derived growth factor, FGF, and bombesin all increased accumulation of c-fos mRNA in these cells (1, 2, 6). Recently, insulin was shown to increase the accumulation of c-fos mRNA in rat H35 hepatoma cells (26).
Evidence is presented in this report suggesting that insulin increases accumulation of c-fos mRNA by stimulating c-fos transcription. The induction of c-fos gene transcription occurred at physiological concentrations of insulin. This is the most rapid stimulatory effect of insulin on gene transcription yet shown, with significant induction of c-fos gene transcription evident within 5 min of insulin addition. The effects of insulin on c-fos gene transcription could be mimicked by serum but not by EGF or FGF. However, in a recently published report, higher concentrations of EGF than were H4 cells were deprived of serum for 24 h and treated with serum, EGF, or FGF. Nuclei were isolated and transcription was measured as described in the legend for Fig. 2 utilized in the present work increased c-fos mRNA levels in hepatoma cells (44).
An increase in c-fos mRNA accumulation in response to high concentrations of insulin was found in a Chinese hamster ovary cell line (CH0.T).
This cell line was transfected with the human insulin receptor gene and expressed 1 million human insulin receptors/cell (45). A sequence in the c-fos 5'flanking region, referred to as the serum-responsive element, was found to be necessary for an induction of the c-fos mRNA by insulin. However, there was no induction of c-fos mRNA by insulin in wild-type CHO cells that did not express this abnormally large number of human insulin receptors. Wildtype CHO cells did contain the normal complement of hamster insulin receptors and had normal insulin responses. The need for an abnormally high membrane concentrations of human insulin receptors to obtain insulin-induced increases of c-fos mRNA in the CH0.T cells questions the physiological significance of this insulin effect. In the present study, a cell line expressing endogenous rat insulin receptors (20,000-30,000/cell (46)) responded to insulin with a rapid increase in the transcription of the c-fos gene.
The rapidity of insulin's effects suggest that few steps intervene between the binding of insulin to H4 cells and the stimulation of c-fos gene transcription. Additionally, the mechanism(s) by which insulin regulates transcription of this gene must be extremely quick since gene expression is induced as rapidly as can be measured. The regulation of this gene by physiological concentrations of insulin in cells containing normal levels of endogenous insulin receptors and the presumed involvement of transcriptional regulatory proteins make the regulation of this gene an interesting model in which to study the role of insulin in the control of gene expression and cell growth.